Non-alcoholic fatty liver disease (NAFLD) is a prevalent metabolic disorder characterized by excessive fat accumulation in the liver, potentially progressing to non-alcoholic steatohepatitis (NASH), cirrhosis, and hepatocellular carcinoma. While dietary imbalances in macronutrients are implicated, the precise roles of other nutrients, including amino acids and nucleobases, remain unclear. Previous research has indicated that low-arginine diets induce fatty liver development in rats, potentially due to altered nucleotide metabolism and increased orotic acid biosynthesis. However, the mechanisms linking altered nucleotide metabolism to hepatic lipid metabolism are poorly understood. This study aimed to investigate the role of serum amino acids in hepatic lipid accumulation using a cell-culture model and non-linear machine learning analysis. The researchers also examined the relationship between nucleobases, serum amino acid profiles, and lipid accumulation in the liver. A previous study suggested a correlation between serum amino acid profiles and hepatic TAG levels; however, causal relationships remained undetermined. This research aimed to clarify whether changes in serum amino acid profiles are a cause or consequence of hepatic lipid accumulation, shedding light on the potential of serum amino acid profiles as diagnostic markers and therapeutic targets for NAFLD.

Numerous studies have shown that dietary imbalances, particularly involving macronutrients and amino acids, significantly affect hepatic lipid metabolism. A low-protein/amino acid diet and a low-arginine diet have been shown to cause fatty liver development. However, the relationship between dietary arginine intake and hepatic TAG levels is not straightforward, and previous work suggested that serum amino acid profiles are more closely associated with hepatic lipid metabolism than dietary intake. A mathematical non-linear analysis previously demonstrated a strong correlation between comprehensive serum amino acid profiles and hepatic TAG levels in rats. Further, many studies suggest that serum amino acid profiles can serve as diagnostic markers for various physiological disorders, including obesity, diabetes, cancer, and NAFLD. While correlations have been established, causality remained elusive. This study addresses this gap by investigating the causal relationship between serum amino acids and hepatic lipid accumulation.

The study used a multi-pronged approach. First, a cell culture model was employed using H4IIE rat hepatoma cells cultured in a medium mimicking the serum amino acid profile of low-arginine (ΔArg) diet-fed rats. This allowed researchers to assess the direct effects of the altered amino acid profile on TAG accumulation in hepatocytes. In parallel, rat models were used, feeding six-week-old male Wistar rats control diets or ΔArg diets with or without adenine, guanine, adenosine, AMP, or IMP supplementation. Hepatic TAG levels were measured to assess the impact of these interventions. The effect of adenine on hepatic TAG secretion was also evaluated using a tyloxapol injection assay, which inhibits lipoprotein lipase and allows for the measurement of hepatic TAG secretion. Liver samples were analyzed for purine metabolite levels (adenine, guanine, adenosine, AMP, IMP) using LC-MS/MS to determine if changes in purine metabolism explained the effects of adenine supplementation. Additionally, the impact of dietary IMP supplementation and intravenous IMP injection was investigated. A multi-layer perceptron (MLP) analysis was performed using previously collected data of serum amino acid concentrations and hepatic TAG levels from rats fed various diets. This machine learning model was then used to predict hepatic TAG levels based on serum amino acid profiles from rats in the current study. Finally, further dietary interventions were conducted with varying methionine, histidine, and BCAA levels in the ΔArg diet to determine causal effects. Serum and liver samples were collected for amino acid profiling using LC-MS/MS. Statistical analyses included Student's t-tests and one-way ANOVA with Tukey-Kramer post-hoc tests.

Culturing hepatocytes in a medium mimicking the serum amino acid profile of ΔArg diet-fed rats led to increased TAG accumulation, demonstrating a direct effect of the altered amino acid profile on hepatocytes. Dietary adenine supplementation completely abolished hepatic TAG accumulation in rats fed a ΔArg diet. This effect was specific to adenine, as other purine metabolites did not have the same effect. Hepatic purine metabolism did not fully explain the mechanism of fatty liver prevention by adenine. MLP analysis accurately predicted the suppression of TAG accumulation using only serum amino acid profiles, suggesting that adenine alters serum amino acid profiles. Comparison of serum amino acid profiles revealed that the AArg + adenine group differed significantly from other groups. Methionine, histidine, and BCAA levels were lower in the AArg + adenine group. Reducing methionine or BCAA levels in the ΔArg diet significantly reduced hepatic TAG accumulation, while histidine deficiency had minimal impact. These findings show a causal link between specific serum amino acid profiles and hepatic TAG accumulation, with adenine intervention altering this profile to reduce TAG accumulation.

The study provides compelling evidence that changes in the comprehensive serum amino acid profile are causally linked to hepatic lipid metabolism regulation. Dietary adenine supplementation influences hepatic TAG accumulation not by directly affecting purine metabolism but by modifying the serum amino acid profile. This novel finding suggests a “metabolic regulatory amino acid signal” driven by the overall serum amino acid profile rather than individual amino acids. The data suggest that methionine and BCAAs are crucial components of this signal, but further research is needed to understand how these signals are recognized by hepatocytes and the specific mechanisms involved in the regulation of lipid metabolism. While the study focuses primarily on amino acid signals, it acknowledges exceptions like hepatitis C virus-induced NAFLD, which may not be directly linked to serum amino acid profiles. This research offers a new perspective on NAFLD pathophysiology, potentially leading to novel diagnostic methods and treatment strategies.

This study demonstrates a previously unknown mechanism of hepatic lipid metabolism regulation by serum amino acid profiles. Dietary adenine supplementation effectively reduces hepatic TAG accumulation by altering this profile, particularly affecting methionine and BCAA levels. This discovery provides valuable insights into NAFLD pathophysiology and opens avenues for developing new diagnostic tools and therapeutic strategies. Future research should investigate the specific receptors and signaling pathways involved in this amino acid-mediated regulation of hepatic lipid metabolism.

While this study provides strong evidence for the role of serum amino acid profiles in hepatic lipid metabolism, further investigation is needed to fully elucidate the mechanisms involved. The study focused primarily on a rat model, and it is important to confirm these findings in human studies. The specific combinations of amino acids necessary for the signal transduction and the cellular mechanisms by which these signals are recognized and transduced into metabolic changes warrant further research. The study used a relatively limited number of rats; larger-scale studies would strengthen the findings.